The invention relates to the use of nucleic acids encoding the kinesin KSP and their gene products to identity modulators of cell proliferation and their use in diagnosis, prognosis and treatment of cell proliferation states and disorders, for example cancer.
Cancer is the second-leading cause of death in industrialized nations. Effective therapeutics include the taxanes and vinca alkyloids, agents which act on microtubules. Microtubules are the primary structural element of the mitotic spindle. The mitotic spindle is responsible for distribution of replicate copies of the genome to each of the two daughter cells that result from cell division. It is presumed that it is the disruption of the mitotic spindle by these drugs that results in inhibition of cancer cell division, and also induction of cancer cell death. However, microtubules also form other types of cellular structures, including tracks for intracellular transport in nerve processes. Therefore, the taxanes have side effects that limit their usefulness. Furthermore, taxanes and vinca alkaloids specifically target microtubule polymerization dynamics. There are additional dynamics of the mitotic spindle that these compounds do not target.
Therefore, it is desirable to identify agents and compositions which are specific and therapeutically effective against cancer. It is further desirable to identify agents and compositions which have a novel mechanism of action. It is further desirable to provide methods of diagnosis of hyper or hypo proliferation disorders. Additionally, it is desirable to identify agents and compositions which modulate cell proliferation. Cell proliferation modulation is desirable in a number of cases as discussed below, for example, for treatment of any hyper or hypo proliferation disorder, wound healing, transplantation procedures and for use in the agricultural arena. It is thus desirable to provide such methods of treatment. Moreover, it is desirable to provide assays to quickly identify such agents and compositions.
Provided herein are assays for screening for bioactive agents which affect cell proliferation. Also provided herein are methods of diagnosing proliferation states in a cell which are useful for identifying cell proliferation disorders such as cancer. Also provided are methods of prognosis and methods of treatment including treatment for cancer. As is further described below, a number of compositions and methods are provided.
In one aspect, a method of screening drug candidates is provided. In one embodiment, said method comprises providing a cell that expresses recombinant human KSP or a fragment thereof and adding a drug candidate to said cell under conditions where the drug candidate is taken up by the cell. The method further includes determining the effect of said drug candidate on the bioactivity of said recombinant human KSP. The bioactivity of recombinant human KSP, or particularly the changes in the presence of a drug candidate, can be determined by assays such as those for determining cellular proliferation, cellular viability, and cellular morphology. In a further aspect of the invention, any changes in bioactivity of recombinant human KSP can be determined by assays for determining changes in the mitotic spindle, particularly inhibition of mitosis, and ATP hydrolysis. The methods herein may also determine the bioactivity of recombinant human KSP in the presence and absence of candidate agents by performing assays determining the effect on apoptosis and necrosis.
The methods provided herein can be performed on single individual cells or a population of cells. The cell can be any kind of cell including but not limited to a lymphocyte, cancer cell or an endothelial cell. In one aspect, wherein cancer cells are utilized, cancer growth or inhibition can be determined, and wherein endothelial cells are utilized, angiogenesis or inhibition thereof can be determined.
In another aspect of the invention, a method of screening for a bioactive agent capable of binding to a cellular proliferation protein is provided. Preferably, the cellular proliferation protein is human KSP or a fragment thereof. In one embodiment, said method comprises combining said cellular proliferation protein and a candidate bioactive agent, wherein said candidate bioactive agent is an exogenous agent, and determining the binding of said candidate agent to said cellular proliferation protein.
In a further aspect herein, a method of screening for a candidate protein capable of binding to a cellular proliferation protein, wherein said cellular proliferation protein is KSP or a fragment thereof, is provided. In a preferred method, said method comprises combining a nucleic acid encoding said cellular proliferation protein and a nucleic acid encoding a candidate protein, wherein an identifiable marker is expressed wherein said candidate protein binds to said cellular proliferation protein.
Also provided herein is a method for screening for a bioactive agent capable of interfering with the binding of a cellular proliferation protein, wherein said cellular proliferation protein is KSP or a fragment thereof, and an antibody which binds to said cellular proliferation protein. In one embodiment, the method comprises combining a cellular proliferation protein, wherein said cellular proliferation protein is KSP or fragment thereof, a candidate bioactive agent and an antibody which binds to said cellular proliferation protein and determining the binding of said cellular proliferation protein and said antibody.
In a further aspect of the invention herein, a method for screening for a bioactive agent capable of modulating the activity of a cellular proliferation protein, wherein said cellular proliferation protein is human KSP or a fragment thereof, is provided. In one aspect, said method comprises combining said cellular proliferation protein and a candidate bioactive agent, wherein said candidate bioactive agent is an exogenous agent, and determining the effect of said candidate agent on the activity of said cellular proliferation protein.
Also provided herein is a method of screening drug candidates comprising providing a cell that expresses KSP, adding a drug candidate to said cell, and determining the effect of said drug candidate on the expression of KSP. In a further aspect the method includes comparing the level of expression in the absence of said drug candidate to the level of expression in the presence of said drug candidate, wherein the concentration of said drug candidate can vary when present, and wherein said comparison can occur after addition or removal of the drug candidate. In a preferred embodiment, the expression of said KSP is decreased as a result of the introduction of the drug candidate. Preferably, the cell utilized is a tumor cell.
In a further aspect, a method of evaluating the effect of a candidate drug on cellular proliferation (a candidate cellular proliferation drug) is provided which comprises administering said drug to a patient, removing a cell sample from said patient, and determining the expression profile of said cell, wherein said expression profile includes a KSP gene. In another aspect, the method includes comparing said expression profile to an expression profile of a healthy individual.
In another aspect herein, a method of diagnosing a hyper-proliferative disorder in an individual is provided herein comprising determining the level of expression a KSP gene in an individual and comparing said level to a standard or control level of expression, wherein an increase indicates that the individual has a hyper-proliferative disorder, such as, but not limited to, cancer.
Also provided herein is a method of evaluating the effect of a candidate cellular proliferation drug comprising administering said drug to a patient wherein said patient has cancer and has been identified as expressing KSP at a level higher than an individual not having cancer, removing a cell sample from said patient, and determining the effect on KSP activity, wherein said KSP activity is mitosis.
In the methods provided herein, the cells can come from a variety of sources. For example, samples can be from, but are not limited to, a blood sample, a urine sample, a buccal sample, a PAP smear, cerebral spinal fluid, and any tissue including, breast tissue, lung tissue and colon tissue. In one embodiment, the patient has cancer.
Also provided herein is a method for inhibiting cellular proliferation, said method comprising administering to a cell a composition comprising an antibody to KSP, wherein said antibody is conjugated to a ligand. In one aspect, the ligand of the antibody is tumor cell specific. In another aspect, the ligand facilitates said antibody entry to said cell. Moreover, the antibody can be a humanized antibody. The methods of inhibition can be performed in vitro on cells or in vivo on an individual. In one embodiment, the cells are cancerous. In a further embodiment, the individual has cancer. Another method of inhibiting cellular proliferation in a cell or individual is provided herein which comprises administering to a cell or individual a composition comprising antisense molecules to KSP.
In yet another embodiment herein, a method for inhibiting cellular proliferation is provided which comprises administering to a cell a composition comprising an inhibitor of KSP. In one embodiment the inhibitor is of human KSP or a fragment thereof. In one embodiment, the inhibitor is specific to human KSP. In one embodiment, KSP inhibitors are any agent which disrupts or inhibits KSP activity as further described herein. In one aspect of the invention, the inhibitor of KSP is a small molecule as further defined herein. Generally, small molecules have a molecular weight of between 50 kD and 2000 kD, and in some cases, less than 1500 kD, or less than 1000 kD, or less than 500 kD. Examples of KSP inhibitors include but are not limited to small molecules, ribozymes, antisense molecules, and antibodies. KSP inhibitors arc further described herein and in the application filed Oct. 27, 1999, entitled Methods and Compositions Utilizing Quinazolinones (U.S. Ser. No. 60/198,253, named inventor Jeffrey T. Finer), incorporated by reference in its entirety. The composition which is administered to a cell further comprises an acceptable pharmaceutical carrier in one embodiment. The composition can have a variety of formulations, including, but not limited to those for parental, oral or topical administration.
The methods of inhibiting cellular proliferation can be performed in vitro or in vivo. More particularly, the compositions can be administered to cells in vitro or in an individual. The individual may have a disease or be at risk for disease. Disease states which can be treated by the methods herein are further described below. In one case, the individual has cancer or is at risk for restenosis. The cell can be any cell, preferably a cancer cell. Other preferred cell types include but are not limited to endothelial cells and metastatic cancer cells. In one embodiment, the method of inhibiting by the KSP inhibitor is by disruption of mitosis or induction of apoptosis.
In a further aspect of the invention, a biochip comprising a nucleic acid segment from KSP, wherein said biochip comprises fewer than 1000 nucleic acid probes, is provided. Methods of screening and diagnosing conditions with said biochip are also provided herein.
Other aspects of the invention will become apparent to the skilled artisan by the following description of the invention.